This invention relates to nucleic acid and amino acid sequences of human proteinase molecules and to the use of these sequences in the diagnosis and treatment of cancer and immune disorders.
Proteolytic processing is an essential component of normal cell growth, differentiation, remodeling, and homeostasis. The cleavage of peptide bonds within cells is necessary for the maturation of precursor proteins to their active form, the removal of signal sequences from targeted proteins, the degradation of incorrectly folded proteins, and the controlled turnover of peptides within the cell.
Proteases participate in apoptosis, antigen presentation, inflammation, tissue remodeling during embryonic development, wound healing, and normal growth. They are necessary components of bacterial, parasitic, and viral invasion and replication within a host. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure (Beynon and Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York N.Y., pp. 1-5).
The serine proteases (SPs) are a large family of proteolytic enzymes that include the digestive enzymes, trypsin and chymotrypsin; components of the complement cascade and of the blood-clotting cascade; and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. SPs are so named because of the presence of a serine residue found in the active catalytic site for protein cleavage. The active site of all SP is composed of a triad of residues including the aforementioned serine, an aspartate, and a histidine residue. SPs have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases which cleave after arginine or lysine; aspases which cleave after aspartate; chymases which cleave after phenylalanine or leucine; metases which cleavage after methionine; and serases which cleave after serine.
The SPs are secretory proteins containing N-terminal signal peptides which export the immature protein across the endoplasmic reticulum prior to cleavage (von Heijne (1986) Nuc Acid Res 14:5683-5690). Differences in these signal sequences provide one means of distinguishing individual SPs. Some SPs, particularly the digestive enzymes, exist as inactive precursors or preproenzymes and contain a leader or activation peptide on the C-terminal side of the signal peptide. This activation peptide may be 2-12 amino acids in length and extend from the cleavage site of the signal peptide to the N-terminus of the active, mature protein. Cleavage of this sequence activates the enzyme. This sequence varies in different SPs according to the biochemical pathway and/or its substrate (Zunino et al. (1990) J Immunol 144:2001-2009; Sayers et al. (1994) J Immunol 152:2289-2297).
Cysteine proteases are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian cysteine proteases include lysosomal cathepsins and cytosolic calcium activated proteases, cilpains. Cysteine proteases are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and in their protective role secrete various molecules to repair damaged tissue. These cells may overproduce the same molecules and cause tissue destruction in certain disorders. In autoimmune diseases such as rheumatoid arthritis, the secretion of the cysteine protease, cathepsin C, degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. The cathepsin family of lysosomal proteases includes the cysteine proteases; cathepsins B, H, K, L, O2, and S; and the aspartyl proteases; cathepsins D and E. Various members of this endosomal protease family are differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue distribution while others, such as cathepsin L, are found only in monocytes, macrophages, and other cells of the immune system.
Abnormal regulation and expression of cathepsins has been implicated in various inflammatory disease states. In cells isolated from inflamed synovia, the mRNA for stromelysin, cytokines, TIMP-1, cathepsin, gelatinase, and other molecules is preferentially expressed. Expression of cathepsins L and D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbate the destruction of the rheumatoid synovium (Keyszer (1995) Arthritis Rheum 38:976-984). The increased expression and differential regulation of the cathepsins is linked to the metastatic potential of a variety of cancers and may be of therapeutic and prognostic interest (Chambers et al. (1993) Crit Rev Oncog 4:95-114).
Cysteine proteases are characterized by a catalytic domain containing a triad of amino acid residues similar to that found in serine proteases. A cysteine replaces the active serine residue. Catalysis proceeds via a thiol ester intermediate and is facilitated by the side chains of the adjacent histidine and aspartate residues.
Aspartic proteases include bacterial penicillopepsin, mammalian pepsin, renin, chymosin, cathepsins D and E, and certain fungal proteases. The characteristic active site residues of aspartic proteases are a pair of aspartic acid residues, such as asp33 and asp213 in penicillopepsin. Aspartic proteases are also called acid proteases because the optimum pH for activity is between 2 and 3. In this pH range, only one of the aspartate residues is ionized. A potent inhibitor of aspartic proteases is the hexapeptide, pepstatin, which in the transition state resembles a normal substrate of the enzyme.
Metalloproteases use zinc as an active site component and are most notably represented in mammals by the exopeptidases, carboxypeptidase A and B, and the matrix metalloproteases, collagenase, gelatinase, and stromelysin. Carboxypeptidases A and B are exopeptidases of similar structure and active sites. Carboxypeptidase A, like chymotrypsin, prefers hydrophobic C-terminal aromatic and aliphatic side chains, whereas carboxypeptidase B is directed toward basic arginine and lysine residues. The matrix-metalloproteases are secreted by connective tissue cells and play an important role in the maintenance and function of the basement membrane and extracellular matrix. A naturally occurring inhibitor of metalloproteases, tissue inhibitor of metalloproteases (TIMP), has been shown to prevent the invasion of tumor cells through basement membrane, in vitro, indicating the importance of these enzymes in cell invasion processes such as tumor metastasis and the inflammatory response (Mignatti et al. (1986) Cell 47:487-498).
Protease inhibitors play a major role in the regulation of the activity and effect of proteases. They have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy (1991) Agents Actions Suppl 35:69-76). In particular, low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, seem to be correlated with malignant progression of tumors (Calkins et al. (1995) Biol Biochem Hoppe Seyler 376:71-80). The balance between levels of cysteine proteases and their inhibitors is also significant in the development of disorders. Specifically, increases in cysteine protease levels, when accompanied by reductions in inhibitor activity, are correlated with increased malignant properties of tumor cells and the pathology of arthritis and immunological diseases in humans.
The discovery of new human proteinase molecules and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis and treatment of cancer and immune disorders.
The invention features purified polypeptides, human proteinase molecules, referred to collectively as xe2x80x9cHPRMxe2x80x9d and individually as xe2x80x9cHPRM-1xe2x80x9d; xe2x80x9cHPRM-2xe2x80x9d, and xe2x80x9cHPRM-3xe2x80x9d. In one embodiment, the purified polypeptide, HPRM, comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and fragments of SEQ ID NOs: 1-3.
The invention includes a purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NOs: 1-3 or fragments thereof.
The invention provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof. The invention also includes an isolated variant having at least 90% sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof.
The invention also provides an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID.NO:5, and SEQ ID NO:6 and fragments and complements of SEQ ID NOs:4-6. The invention includes a variant having at least 90% sequence identity to the polynucleotide selected from the group consisting of SEQ ID NOs:4-6 and complements and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof. In another aspect, the expression vector is contained within a host cell. The invention still further provides a the method for using a polynulceotide to produce a polypeptide comprising culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions for the expression of the polypeptide and recovering the polypeptide from the host cell culture.
The invention yet still further provides a method for using a polynucleotide to detect a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NOs:1-3 in a sample comprising hybridizing the polynucleotide or the complement thereof to at least one nucleic acid in the sample, thereby forming a hybridization complex and detecting the hybridization complex, wherein the presence of the hybridization complex indicates the expression of the nucleic acid in the sample. In one aspect, the nucleic acids of the sample are amplified prior to hybridization.
The invention additionally provides a method of using a polynucleotide to screen a plurality of molecules to identify a molecule which specifically binds the polynucleotide comprising combining the polynucleotide with the plurality of molecules under conditions to allow specific binding and detecting specific binding, thereby identifying a molecule which specifically binds the polynucleotide. In one aspect, the molecule is selected from DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, and proteins.
The method provides purified polypeptides comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and fragments thereof. In one aspect, a biologically active fragment of the polypeptide is selected from residues D132-F144 of SEQ ID NO:1, residues C34-C415 of SEQ ID NO:2 and residues V93-V104 of SEQ ID NO:3.
The invention also provides a method for using a polypeptide to screen a plurality of molecules to identify a molecule which specifically binds the polypeptide comprising combining the polypeptide with the plurality of molecules under conditions to allow specific binding and detecting specific binding, thereby identifying a molecule which specifically binds the polypeptide. In one aspect, the molecules are selected from agonists, antagonists, antibodies, DNA molecules, RNA molecules, peptide nucleic acids, immunoglobulins, inhibitors, drug compounds, peptides, and pharmaceutical agents.
The invention further provides a method of using a polypeptide to purify a molecule which specifically binds the polypeptide from a sample comprising combining the polypeptide with a sample under conditions to allow specific binding, recovering the bound polypeptide, and separating the molecule from the polypeptide, thereby obtaining the purified molecule.
The invention still further provides a method for using a polypeptide to produce an antibody, comprising immunizing an animal with the polypeptide under conditions to elicit an antibody response and isolating antibodies which bind specifically to the polypeptide.
The invention yet further provides a method for using a polypeptide to purify an antibody which specifically binds the polypeptide comprising combining the polypeptide with a plurality of antibodies under conditions allow specific binding, recovering the bound polypeptide, and separating the antibody from the polypeptide, thereby obtaining antibody which specifically binds the polypeptide. In one tin aspect, the antibodies are selected from polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies; Fab fragments, Fv fragments, and F(abxe2x80x2)2 fragments.
The invention additionally provides a purified antibody which specifically binds the polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof.
The invention provides compositions comprising an isolated polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof and reporter molecule or a purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof and a pharmaceutical carrier.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof.
The invention further provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3 and fragments thereof.